The immediate objectives of this competing grant renewal are to understand the biochemical and genetic regulatory mechanisms which govern transcription of trypanosome protein-encoding genes and the post-transcriptional processing of pre- mRNAs by trans splicing. They expect these studies to provide the fundamental information necessary to address their long range goal: understanding the biochemical and molecular events which regulate gene expression in trypanosomes. The expression of alternative VSG genes during growth in the mammal permits the parasite to evade clearance by host immune mechanisms and has confounded strategies for controlling African trypanosomiasis through vaccination. The investigators will address the unique processes associated with pre- mRNA transcription in trypanosomes through molecular genetic and biochemical crosslinking experiments. They will identify and characterize in vivo transcription complexes, resolving the basic issue of which RNA polymerase (RNAP) is responsible for a- ammunition resistant pre-mRNA transcription. Preliminary evidence for in vitro transcription initiation of the procyclic surface antigen gene PARP and of rRNA genes will be pursued. Transcription initiation will be characterized biochemically and with mutant promoter elements previously characterized in vivo. They will seek to identify and characterize an a- ammunition sensitive RNAP II promoter using a new extrachromosomal vector and selective and nonselective strategies for promoter isolation. Trypanosome polycistronic pre-mRNA transcripts require RNA processing by trans splicing, an RNA pathway which was first discovered in T. brucei. They will continue their ongoing analysis of the components of trans splicing by in vivo crosslinking, characterizing the RNA-RNA and RNA- protein interactions of trans splicing. They have defined two new small RNA species in this manner and will characterized these with respect to their potential role in RNA processing. Structural and sequence requirements of a trans splice acceptor will be addressed by molecular genetics. Synthetic acceptor constructs, dependent upon branch point sequences for expression, will also be used to examine the molecular basis of branchpoint selection and to develop genetic complementation protocols for T. brucei. Finally, they will use these aggregate approaches and information to reconstitute trans splicing in vitro. If successful, a trypanosome in vitro trans-splicing extract will be the first developed in so primitive a eukaryotic system. The only other in vitro trans splicing system is derived from Ascaris, which also uses cis splicing in pre-mRNA maturation. An in vitro trypanosome trans splicing system will allow molecular and biochemical comparisons between cis and trans splicing. RNA processing in trypanosomes by potentially novel processes will contribute not only to our fundamental knowledge of transcription and trans splicing, but should also reveal important differences in the parasite and host which may lead to strategies of disease intervention and control.